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1st International Virtual Conference on Pure Science IOP Publishing
Journal of Physics: Conference Series 1664 (2020) 012084 doi:10.1088/1742-6596/1664/1/012084

Spectrophotometric Determination of tetracycline hydrochloride Using

2,4 –dinitrophenyl hydrazine as Coupling Reagent

Roaa M. Khaleel1 and Dawooh H. Mohammed2

1,2
Chemistry Department / /Education College for Girls/ Mosul University

*e-mail:Roaa.mohanad.1989@gmail.com1 , Dr.alhaboo@uomosul.edu.iq2

ABTRACT
Simple, highly sensitive and accurate spectrophotometric method has been developed to
determination of tetracycline hydrochloride in aqueous solution. The proposed method is based on the
coupling of tetracycline hydrochloride with 2,4-dinitrophenylhydrazine (2,4-DNPH) in the presence
of potassium periodate to form an intense orange color dye at 360nm. Beer's law is obeyed in the
concentration range 0.1-9 μg/ml with the molar absorptivity of 1.262 ×104 liter.mol-1 .cm-1,with limit
of detection (LOD) of 0.0123μg/ml and limit of quantification (LOQ) of 0.0412μg/ml while the RSD
value of 0.184 – 0.467 % depending on the concentration. The proposed method was performed
successfully to the determination and analysis of tetracycline hydrochloride in pharmaceutical
formulations with average recovery of 100.23 % .

Key words: Spectrophotometric, Tetracycline hydrochloride, 2,4-DNPH reagent.

1. Introduction

Tetracycline hydrochloride is considered a member of the family of tetracycline hydrochloride’s, which

offers biomass interpreters on a wide spectrum. It has been frequently used in treating negative Gram and positive
Gram and bacteria. It is also considered as one of the least toxic drugs and the best family of medicines. The
molecular form of the Tetracycline hydrochloride is C22H24N2O8.HCl, while the molecular weight is 480.93 g/mol
[1]. Fig.(1) depicts the structure formula of Tetracycline:

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Published under licence by IOP Publishing Ltd 1
1st International Virtual Conference on Pure Science IOP Publishing
Journal of Physics: Conference Series 1664 (2020) 012084 doi:10.1088/1742-6596/1664/1/012084

Fig.(1): Chemical structure of Tetracycline hydrochloride

tetracycline hydrochloride is mainly effective for bacteriostatic treatment and is used in the field of human and
veterinary medicine by linking it with the ribosome 30S. It prevents the arrival of Amino acyl tRNA to future sites
in the mRNA complex [2] and exerts its effect against microbes. The practical mechanism is to inhibit the protein
industry in bacteria cells through blocking the access of amino acids to the ribosome. The main working area of
tetracycline hydrochloride is in the protein industry. It has been found that tetracycline hydrochloride can be
disseminated through the outer cell membrane of Gram-negative bacteria [3]. The misuse or overuse of tetracycline
hydrochloride yields the formation of bacteria resistant, which reduces its usage [4]. Tetracycline hydrochloride is
taken orally and absorbed in the duodenum and rapidly spread in body fluids and tissues. It is concentrated in the
bile and excreted through the kidneys and its absorption can be further reduced by milk alkaline materials and
products, aluminum salts, magnesium and calcium. It is occasionally available in ointments to treat skin infections
and acne [5]. The determination methods of tetracycline hydrochloride are; spectrophotometric methods [6-10],
Chromatographic methods [11-17] and Electrical methods [18-20].

2.Experimental

2.1 Apparatus

x Spectrophotometric measurements were performed using a dual-beam spectrophotometer: UV-VIS 1800

x spectrophotometer, Japan Shimadzu with quartz type cells 1 cm in all absorption measurements.
x The weighing was performed using the balance type of: artorius BL 201 S

2.2 Reagents

All the used chemicals and analytical reagents were of a high grade of purity and available from
commercial sources in the local market and are used directly. Distilled water was used for all dilutions of reagents
and samples.

2.3 Material solutions

x Standard tetracycline hydrochloride solution (100 mg / ml): This solution is dissolved 0.01 g of the pure
material in distilled water, solution is transferd into a 100ml volumetric flask.
x potassium periodate (5×10-3M): This solution is dissolved 0.1150 g of pure material in 3 ml of dilute
sulfuric acid and complete the volume using distilled water in a 100 ml volumetric flask.
x 2,4 –dinitrophenyl hydrazine solution (2×10-3M): This solution is prepared by dissolving 0.1981 g of
material in 5 ml of dilute sulfuric acid and completes the volume with distilled water in a 100 ml
volumetric flask.
x Surfactant: the SDS and CTAB surfaces were papered at 0.1% concentration is dissolved 0.1g of it in
100ml of distilled water. Triton x-100 solution is prepared at a concentration 1% is dissolved one gram of
it in 100ml of distilled water

3.Procedure for pharmaceutical preparations

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1st International Virtual Conference on Pure Science IOP Publishing
Journal of Physics: Conference Series 1664 (2020) 012084 doi:10.1088/1742-6596/1664/1/012084

10 capsules were grinded well and a certain portion of the final powder was accurately weighted to give an
equivalent to about 250 mg of tetracycline hydrochloride was dissolved distilled water . The prepared solution
-1
transferred to 100 ml volumetric flask and made up to the mark with distilled water forming a solution of 100μg ml
concentration . The solution was filtered by using a filter paper to avoid any suspended particles.

4. Results and discussion

1 ml of 2,4 –dinitrophenyl hydrazine solution (2×10-3M) were added into a series of 25ml calibrated flask
and 2 ml of potassium periodate (5×10-3M) followed by the addition of increasing volumes of (100μgmlˉ¹)
tetracycline hydrochloride and followed by 1 ml of (0.1%) Triton X-100. and wait for three minutes for the purpose
of completing the oxidation ,then absorbance of all solutions was measured at a wavelength of 360 nm versus their
blank solutions.

4.1 Study of the Optimum Reaction Conditions:

The spectrophotometric properties of the colored product as well as the different experimental parameters
that affect the color development and its stability were carefully studied and optimized by using 1 ml of tetracycline
hydrochloride at a concentration of 100 mg / ml in a final volume of 25 ml (with a final concentration of 4 mg / ml).

4.2 Effect of amount the reagent 2,4 –dinitrophenyl hydrazine

The concentrations and volume of different reagents that affect the 2,4–dinitrophenyl hydrazine reactions
were also carefully studied and optimized by increasing volumes 0.25-2 ml from 2,4-di-nitrophenyl hydrazine 2 x
10-3 molar to a volume of 25 ml containers, which contains 1 ml of tetracycline hydrochloride solution (100 mg / ml)
and 1 ml from a potassium periodate solution(5 × 10-3 molar) and diluted to the mark with distilled water. The
experimental results are presented in Table 1.

Table 1: Effect of amount 2,4 –dinitrophenyl hydrazine

ml of 2,4 Absorbance / min standing time

Dinitrophenyl
hydrazine 2×10-3M
1 3 5 7 10
0.25 1.249 1.247 1.248 1.248 1.247
0.5 1.258 1.257 1.257 1.256 1.255
1 1.266 1.266 1.265 1.266 1.266
1.5 1.227 1.228 1.228 1.226 1.225
2 1.198 1.196 1.193 1.191 1.188

4.3 Effect of amount of the oxidization agent (potassium periodate) :

The reaction of oxidization amount to reagent was studied. The absorbent was measured at different
periods of time. The results are shown in Table 2.

Table 2: Effect of oxidization amount

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1st International Virtual Conference on Pure Science IOP Publishing
Journal of Physics: Conference Series 1664 (2020) 012084 doi:10.1088/1742-6596/1664/1/012084

ml of 5×10-3 M Absorbance / min standing time

Potassium
periodate
1 3 5 7 10

1 1.263 1.264 1.265 1.265 1.264

1.5 1.269 1.268 1.267 1.267 1.266
2 1.275 1.274 1.275 1.275 1.274
2.5 1.249 1.250 1.250 1.251 1.247
3 1.231 1.232 1.234 1.235 1.230
4.4 Effect of surfactants:

The use of different surface tension, cationic, anionic and neutral materials as a result of the instability of
the dye formed with a view to show the effect on the intensity and stability of the colored product .The results are
shown in Table 3.

Table 3: Effect of surface tensioning agents on absorbing the resulting dye

Surfactant Absorbance / ml of surfactant used

0.5 1.0 1.5 2 2.5

SDS (0.1%) 1.225 1.227 1.228 1.229 1.226

Triton X-100 (1%) 1.275 1.277 1.272 1.270 1.268

CTAB (0.1%) 1.249 1.250 1.253 1.258 1.256

4.5 Effect of oxidation time:

The time required to complete the oxidation was studied by taking a set of 25 ml volumetric containers
containing 1 ml of tetracycline hydrochloride solution at a concentration of 100 mg / ml, 1 ml of reagent solution 2,4
dinitrophenyl hydrazine 2 x 10-3 molar, 2ml of potassium periodate 5 x 10-3 molar and added 1 ml of Triton-X100
at a concentration of 2%. Finally, discard the solutions for different time periods, then dilute it with distilled water
up to the mark and then measure the absorption of the solutions at a wavelength of 360 nm against their blank
solution. The results are shown in Table 4.

Table 4: Effect oxidation time

Tetracycline hydrochloride

Time (Min) Directly 3 5 10 15 20

Absorbance 1.275 1.277 1.274 1.270 1.266 1.261

4.6 Order of addition

In order to choose the best addition sequence for the reactants, a number of different laboratory
experiments were conducted to find out the effect of the addition sequence of solutions. The results shown in Table
5, it explains that the best addition sequence is (Ι), to form the colored product and it was adopted in subsequent
experiments accordingly.

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1st International Virtual Conference on Pure Science IOP Publishing
Journal of Physics: Conference Series 1664 (2020) 012084 doi:10.1088/1742-6596/1664/1/012084

Table 5: Effect of order of addition on absorption

Order of addition
Order number Absorbance

D+R+O+Triton-100 Ι 1.277
O+R+Triton-100+D Π 1.217
D+O+R+Triton-100 Ш 1.266
R+D+O+Trhton-100 IV 1.168
D+R+Triton-100+O V 1.123
R+O+Triton-100+D VI 1.197
-3
R: 2,4 –dinitrophenyl hydrazine (2×10 M)

O: potassium periodate (5×10-3M)

D: Tetracycline hydrochloride (100 mg/ml)

4.7 Final absorption spectra:

The final absorption spectrum for the formed product was drawn up and according to the optimal
conditions, where the absorption spectra graph showed the maximum intensity of absorption at a wavelength of 360
nm while the blank solution gained a weak absorption at this region as shown in Fig.(2).

2.000

A
1.500 B

C
Abs.

1.000

0.500

0.000
300.00 350.00 400.00 450.00 500.00
nm.
Fig.(2): Absorption spectra of 4 μgml- tetracycline hydrochloride measured
[A:sample and blank, B:sample and water, C:blank and water]

4.8 Quantification

Under the optimum conditions, a linear calibration curve is constructed by plotting absorbance versus
concentration. Beer's law is obeyed over the range (0.1-9)µg /ml of the solution Fig.(3).

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1st International Virtual Conference on Pure Science IOP Publishing
Journal of Physics: Conference Series 1664 (2020) 012084 doi:10.1088/1742-6596/1664/1/012084

3
y = 0.2625x + 0.1976
2.5 R² = 0.9994

Absorbance
2
1.5
1
0.5
0
0 2 4 Conc. mg/ml 6 8 10

Fig.(3): Calibration Curve for Tetracycline hydrochloride Determination

4.9 Accuracy and precision

The RSD ratio was calculated for three concentrations 2, 3.6, 6 mg / ml of tetracycline hydrochloride. By
computing the average of six iterations for each and treating according to the optimal conditions, the results were
summarized in Tabl 6. These results tell us that the proposed method provided an efficient accuracy recovery rate
(100.23%), and high precision, while the value of RSD did not exceed 0.467. The results are depicted in Table 6.

Table 6: Accuracy and precision

Concentration of Average
Concentration of
Relative Error
Tetracycline.HCl Tetracycline.HCl Recovery % Recovery % RSD %
%
taken (µg/ml)
found (µg/ml)

2 1.99 99.5 -0.5 r 0.467

3.6 3.604 100.11 100.23 0.11 r 0.259

6 6.065 101.08 1.08 r 0.184

4.10 Nature of the product:

Continuous changes methods (Job method) [21] and molar ratio were applied to study the structural ratio of
the colored product consisting of the coupling of reagent 2,4-di-nitrophenylhydrazine with tetracycline
hydrochloride in the aqueous solution. The concentration of the reagent and tetracycline hydrochloride are 2 × 10-4
molar. The number of solutions was prepared by taking volumes from the pharmaceutical compound and the
reagent. Therefore, the total volume of the two solutions was fixed by 3 milliliters. The rest of the additions were
completed according to the optimal conditions and dissolving with distilled water up to the mark. Absorbance of the
solutions was measured wavelength 360 nm versus its blank solutions, Fig.(4) shows that the ratio was 1:1.

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1st International Virtual Conference on Pure Science IOP Publishing
Journal of Physics: Conference Series 1664 (2020) 012084 doi:10.1088/1742-6596/1664/1/012084

1.5

Abssorbance
1

0.5

0
0 0.2 0.4 0.6 0.8 1
[S]/[S]+[R]

[S]=Tetracycline hydrochloride conc. , [R]=Ragent conc.

Fig.(4): Continuous Change Method for Tetracycline hydrochloride Solution with reagent 2,4-
Diphenyl Hydrazine

In order to know the nature of the resulting dye, the molar ratios method was applied, which included the
addition of increased volumes of reagent solution 2,4-di-nitrophenyl hydrazine at a concentration of 2 x 10-4 mol to a
fixed volume 1 ml of tetracycline hydrochloride solution. The results are shown in Fig.(5). In fact, these results are
in agreement with the job’s method result. The proposed dye for the colored product is shown in Fig.(6).

Fig.(5): plotting the molar ratio of the tetracycline

reaction with
4.2-di-nitrophenylhydrazine.

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1st International Virtual Conference on Pure Science IOP Publishing
Journal of Physics: Conference Series 1664 (2020) 012084 doi:10.1088/1742-6596/1664/1/012084

Fig.(6): Nature of the colored product formed

4.11 Proposed chemical reaction:

The Fig.(7) explains the proposed chemical reaction:

Fig.(7): Proposed chemical reaction:

4.12 Interference effect:

In order to test the efficiency and selectivity of the proposed method, the effect of the presence of some
common pharmaceutical additive such as Sucrose, Fructose, Starch,Sodium chloride and Glucose that are usually
present in dosage forms was studied by adding different amounts of foreign substances to 4 mg of tetracycline
hydrochloride. The result in Table 7, indicates that there were no significant interference produced by the foreign
substances on the proposed procedure.

Table 7: Effect of interference Tetracycline hydrochloride

Recovery% ,of 4 μgml-1of , per

Tetracycline hydrochloride
compounds μgml-1of foreign compounds
added
100 300 500
μgml-1 μgml-1 μgml-1
Sucrose 100.70 100.01 101.33

Fructose 97.96 98.35 98.66

Starch 100.62 100.23 100.93

Sodium chloride 97.24 97.49 97.80

Glucose 98.66 99.06 99.45

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1st International Virtual Conference on Pure Science IOP Publishing
Journal of Physics: Conference Series 1664 (2020) 012084 doi:10.1088/1742-6596/1664/1/012084

4.13 Standared additiom method:

To prove the efficacy of the proposed method and its accuracy in determining that no significant
interference, the standard addition was applied to the pharmaceutical preparation. The method included adding 0.5
and 0.75 ml of the pharmaceutical preparation previously prepared. Fig.(8) and Table 8. reflect standard addition of
tetracycline hydrochloride.

2.5
y = 0.285x + 0.85
2 R² = 0.9991

1.5

B(3 mg/ml)
1
A(2 mg/ ml)
y = 0.285x + 0.55
0.5 R² = 0.9991

0
-4 -2 0 2 4 6
-0.5

Fig.(8): Standard addition of tetracycline hydrochloride

Table8: Determination of tetracycline hydrochloride in pharmaceutical

preparations using the standard addition method

Tetracycline HCL Tetracycline HCL

Type of drug Recovery (%)
Present mg/ml Measured µg/ml

Tetracycline HCL 2 1.99 99.5

Capsule (250)mg 3 3.01 100.33

5. Conclusion

In this work could be concluded the proposed method for determination Tetracycline hydrochloride assay
was simple, precise, accurate, and sensitive. The proposed method can be satisfactorily applied to the analysis of
tetracycline hydrochloride in bulk and pharmaceutical formulations.

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